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Article Effects of Seawater on Production and Content of Engineered Saccharomyces cerevisiae

Yuqi Guo 1 , Shangxian Xie 2,3, Joshua S. Yuan 2,3 and Katy C. Kao 1,*

1 Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA; [email protected] 2 Synthetic and Systems Biology Innovation Hub, Texas A&M University, College Station, TX 77843, USA; [email protected] (S.X.); [email protected] (J.S.Y.) 3 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA * Correspondence: [email protected]

 Received: 8 October 2018; Accepted: 24 December 2018; Published: 1 January 2019 

Abstract: The use of seawater in fermentation can potentially reduce the freshwater burden in the bio-based production of chemicals and fuels. We previously developed a Saccharomyces cerevisiae hyperproducer SM14 capable of accumulating 18 mg g−1 DCW (DCW: dry weight) of β-carotene in rich media (YPD). In this work, the impacts of seawater on the carotenoid production of SM14 were investigated. When using nutrient-reduced media (0.1× YNB) in freshwater the β-carotene production of SM14 was 6.51 ± 0.37 mg g−1 DCW; however in synthetic seawater, the production was increased to 8.67 ± 0.62 mg g−1 DCW. We found that this improvement was partially due to the NaCl present in the synthetic seawater, since supplementation of 0.5 M NaCl in freshwater increased β-carotene production to 11.85 ± 0.77 mg g−1 DCW. The combination of synthetic seawater with higher carbon-to-nitrogen ratio (C:N = 50) further improved the β-carotene production to 10.44 ± 0.35 mg g−1 DCW. We further showed that the carotenoid production improvement in these conditions is related with lipid content and composition. These results demonstrated the benefit of using seawater to improve the production of carotenoids in S. cerevisiae, and have the potential to expand the utilization of seawater.

Keywords: bioproduct; fermentation; biotechnology; seawater; physiology

1. Introduction Carotenoids belong to a large class of pigmented compounds naturally produced by plants, algae, fungi, and bacteria [1–4]. In the native organisms, these compounds serve as natural colorants, photoprotective agents, and antioxidants [1,3,5,6]. Carotenoids also play roles as A precursors, antioxidants, and antimicrobials in [5,7]. Thus, these compounds have potential applications in the food, cosmetics, and health industries [1,6,8]. Currently, most of the carotenoids produced in industry are extracted from natural sources such as plants and algae, or chemically synthesized. However, the structural complexity of most carotenoids make them difficult to be synthesized chemically, and the solvent-based chemical extraction from plants and algae faces challenges due to unpredictable feedstock availability [6,9]. Thus, there have been extensive efforts to engineer microbial hosts for their production [10,11]. In our prior work, we developed a novel evolution engineering approach based on the antioxidant potential of β-carotene and successfully generated carotenoid hyper-producing strains of Saccharomyces cerevisiae [12]. Briefly, crtE, crtYB, and crtI were introduced into the S. cerevisiae strain GSY1136 [13] via the integration vector YIplac211YB/I/E* [10]. To test the hypothesis that production of β-carotene can protect the cells from oxidative stress, the catalase gene CTT1 was deleted to remove the native capacity of yeast for detoxifying hydrogen

Fermentation 2019, 5, 6; doi:10.3390/fermentation5010006 www.mdpi.com/journal/fermentation Fermentation 2019, 5, 6 2 of 9 peroxide. The resulting strain YLH2 (GSY1136 YIplac211YB/I/E* ∆CTT1) was evolved by periodic challenge with hydrogen peroxide, and the evolved isolates exhibiting increased carotenoid production (SM11, SM12, SM13, SM14, SM22, SM24, etc.) were selected based on their darker red color [12]. In subsequent work, we optimized bioreactor conditions for production using freshwater [14]. However, industrial fermentation requires a large amount of water, which generates pressures on freshwater [15]. Using alternative water sources such as unpurified seawater in industrial fermentation will help to reduce freshwater usage in bio-based production of chemicals [16], especially in arid regions of the world that have easy access to saline water sources. Prior work has shown the use of seawater media to be promising for ethanol fermentation by Saccharomyces cerevisiae [17], and seawater media has also been used to improve carotenoid production in Rhodotorula glutinis [18]. Therefore, seawater may also have the potential to improve carotenoid production in engineered Saccharomyces cerevisiae. In this work, we analyzed the impact of using seawater and reduced nutrients on the carotenoid productivity of a hyper-producer (SM14) generated from our prior work [12], and showed that the carotenoid production of the engineered non-native producer can also be improved by using seawater media.

2. Results and Discussion

2.1. Impact of YNB Concentration on β-Carotene Production of SM14

+ 2+ + 2+ −1 2− Seawater contains trace elements (Na , Mg ,K , Ca , Cl ) and SO4 that are present in yeast nitrogen base (YNB) (see Table S1 for comparison). Thus, we hypothesized that using seawater may reduce the requirement of YNB, and compared the β-carotene production of SM14 with different concentrations of YNB (1×, 0.7×, 0.4×, 0.1×) (while keeping the concentration of other constituents the same as described in Materials and Methods) in freshwater and seawater. Results showed that in 1×, 0.7×, or 0.4× YNB, biomass formation and β-carotene concentrations were both lower in seawater than in freshwater, but β-carotene yields were not significantly different between freshwater and seawater (Figure S1). Interestingly, in 0.1× YNB, the biomass formation, β-carotene concentration, and yield in seawater were significantly higher than that in freshwater (p-value < 0.01). Furthermore, the β-carotene yield in seawater in 0.1× YNB was significantly higher than in higher concentrations of YNB (p-value < 0.01), which suggests that SM14 produced and accumulated more carotenoids in seawater under nutrient-reduced conditions. Since the difference in β-carotene production between freshwater and seawater was most obvious with lower YNB concentration, subsequent experiments were conducted with 0.1× YNB.

2.2. Impact of Water Sources on β-Carotene Production of SM14 For nutrient-reduced condition, we first tested the impact of water source on the carotenoid production of SM14 in 20 mL media using either freshwater, 1/3× seawater (water: synthetic seawater = 2:1), or seawater in 125 mL flasks. As show in Figure1a, the growth of SM14 was significantly inhibited in seawater, with a 24 h longer lag phase; however, the culture reached a higher final cell density compare with that in freshwater. Interestingly, no growth inhibition and a higher final cell density were observed in 1/3× seawater. The β-carotene production of strain SM14 in both seawater and 1/3× seawater were significantly higher compared to that in freshwater (Figure1b). The β-carotene yield observed in freshwater peaked at 8.25 ± 0.51 mg g−1 DCW (DCW: dry cell weight) after 72 h. On the other hand, the β-carotene production in seawater and 1/3× seawater continued to increase and reached 13.70 ± 1.29 mg g−1 DCW and 17.37 ± 1.12 mg g−1 DCW, respectively, after 120 h. FermentationFermentationFermentationFermentationFermentationFermentationFermentation 2019 2019, 20195 2019,, 2019x 52019 , ,2019 x20195, ,5 ,x5, 5 ,x, ,, x 55x ,, x 6x 3 of3 of93 3 of39 3of of393 of3 9 of of9 of9 9 9 FermentationFermentationFermentation 2019 20192019, ,,5 55, ,,x xx 333 of ofof 9 99

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Thus, Thus,Thus, it itit is isis not notnot known knownknown at atat this thisthis point pointpoint whether whetherwhether the thethe commonalities commonalitiescommonalities between betweenbetween the thethe osmoticosmoticosmoticosmoticosmoticosmotic osmoticbetweenand and and oxidativeand andoxidativeand and theoxidative oxidative oxidativeoxidative osmoticoxidative stress stress stress stress and stresstolerancestress stresstolerance oxidativetolerance tolerance tolerancetolerance tolerance regulon regulon stressregulon regulon regulonregulon regulonare tolerance are directlyare aredirectly areare directlyare directly regulondirectlydirectly directlyconnected connected connected connected are connectedconnected connected directly with with with withthe with connected with thewith carotenoidthe the carotenoidthethe carotenoidthe carotenoid carotenoidcarotenoid withcarotenoid hyper- thehyper- hyper- carotenoid hyper- hyper-hyper- hyper- osmoticosmoticosmotic andandand oxidativeoxidativeoxidative stressstressstress tolerancetolerancetolerance regulonregulonregulon areareare directlydirectlydirectly connectedconnectedconnected withwithwith thethethe carotenoidcarotenoidcarotenoid hyper-hyper-hyper- producingproducingproducingproducingproducingproducingproducinghyper-producing phenotype phenotype phenotype phenotype phenotype phenotype phenotype in inSM14. phenotype inSM14. in in SM14.in SM14. in SM14. SM14. SM14. in SM14. producingproducingproducing phenotype phenotypephenotype in inin SM14. SM14.SM14.

FigureFigureFigureFigure Figure2.Figure FigureEffect2. Effect2. 2. 2.Effect 2. Effectof Effect2. Effect of NaClEffect ofNaCl of of NaCl ofsupplementationNaCl of NaCl NaClsupplementation NaCl supplementation supplementation supplementation supplementation supplementation on on the on theon growthon onthe thegrowthon onthe the growth the thegrowth growth growthkinetics growth growth kinetics kinetics kinetics kinetics kineticsand kinetics kinetics and βand -caroteneand βand and-carotene βand and β-carotene β -caroteneβ-carotene -caroteneβ -carotene-caroteneproduction production production production production production production production of ofSM14 ofSM14 of of SM14of SM14 of SM14of SM14 SM14 SM14 FigureFigureFigure 2. 2.2. Effect EffectEffect of ofof NaCl NaClNaCl supplementation supplementationsupplementation on onon the thethe growth growthgrowth kinetics kineticskinetics and andand β ββ-carotene-carotene-carotene production productionproduction of ofof SM14 SM14SM14 in in0.1× in0.1×in inYNB0.1×in 0.1× in0.1×YNBin 0.1× 0.1×YNB 0.10.1×media YNB YNB ×mediaYNB YNBYNB YNBmedia media inmedia media infreshwater.mediamedia media infreshwater. in in freshwater.in freshwater. infreshwater.in infreshwater. freshwater.freshwater. freshwater. (a )( agrowth )( agrowth( a)( a()growtha ) growth(( ) (agrowth agrowthkinetics))) growthgrowth growthkinetics kinetics kinetics kinetics kinetics(DCW: kineticskinetics kinetics (DCW: (DCW: (DCW: (DCW: dry(DCW: (DCW:(DCW: (DCW:dry cell dry dry celldry dryweight) celldry dry dry cell weight)cell cell weight) cell cell weight) weight) weight)with weight)weight) with with0 with Mwith 0with Mwith with( 0 0 M(0 M0 M ),(0M0 ( M0.05 M( ),( 0.05( ( ), ), M0.05 ), ),0.05 0.05M ), ),0.05 0.05M 0.05 M M M M M ininin 0.1× 0.1×0.1× YNB YNBYNB media mediamedia in inin freshwater. freshwater.freshwater. ( (a(aa)) )growth growthgrowth kinetics kineticskinetics (DCW: (DCW:(DCW: dry drydry cell cellcell weight) weight)weight) with withwith 0 00 M MM ( (( ), ), ), 0.05 0.050.05 M MM (( ),(( 0.137( ),( 0.137(( ), ), 0.137 ), M ),0.137 0.137 ), ),0.137 M( 0.1370.1370.137 M( M M ),(M M( MM0.2( ),( ( 0.2(( ),M ), 0.2 ), ),M0.2 (),0.2 ), ),0.2 M 0.2 ( 0.2M0.2 M ),(M M ( MM0.3( ), ( 0.3(( ),M ), 0.3 ), ),M0.3 (0.3 ), ),0.30.3 M ( 0.3M0.3 M M ),(M ( MM0.41 ( ),( 0.41(( ), ), M0.41 ), ),0.41 0.410.41M ),( ),0.41 0.41M0.41( M MM )(M ( and MM( )( and (( ) 0.5 )and ) andand )and0.5 and )M ) and0.5and 0.5 M0.5 (0.5 0.5 M M( 0.5M0.5 M ) (M ( NaCl. MM( ) (NaCl. ( )( ) NaCl.NaCl. ) NaCl. )(NaCl. bNaCl. ) )) (NaCl. NaCl.bβ ()-carotene(b bβ( )b() -caroteneb (β)βb )-carotene(β(-carotene )bβ -caroteneβ))-carotene -caroteneβ -carotene-carotene yield ((( ), ), ), 0.137 0.1370.137 M MM ( (( ), ), ), 0.2 0.20.2 M MM ( (( ), ), ), 0.3 0.30.3 M MM ( (( ), ), ), 0.41 0.410.41 M MM ( (( ) ) ) and andand 0.5 0.50.5 M MM ( (( ) ) ) NaCl. NaCl.NaCl. ( (b(bb)) ) β ββ-carotene-carotene-carotene yieldyieldyield (yieldyield yield(yield( ) (and) ( ) ( and(and )( )concentrationand ) concentrationand) andconcentration )and andconcentration concentration concentrationconcentration concentration ( ( () ) after( (after )( ( after )( 72 ) after )72 after) h.after )after72 h.after Error 72 h. 72Error 72 72h. Error h.72 barsh.Errorh. Errorbarsh. Error Error bars areError bars arebars standard barsarebars standardbarsare arestandard areare standard are standard deviations. standardstandard standarddeviations. deviations. deviations. deviations. deviations.deviations. *:deviations. Statistically*: Statistically*: Statistically*: *: *:Statistically *: Statistically *:StatisticallyStatistically significantlyStatistically yieldyieldyield ((( ) ) ) andandand concentrationconcentrationconcentration ((( ) ) ) afterafterafter 727272 h.h.h. ErrorErrorError barsbarsbars areareare standardstandardstandard deviations.deviations.deviations. *:*:*: StatisticallyStatisticallyStatistically significantlysignificantlysignificantlysignificantlysignificantlysignificantlysignificantlydifferentsignificantly different different different fromdifferent different different fromdifferentdifferent thefrom fromthe controlfrom from fromthe controlfromfrom the thecontrol the (nothe control thethecontrol control (no NaCl)control control control(no NaCl) (no (noNaCl) (*(no (no NaCl) p (no(no NaCl)-value (*NaCl) NaCl) p (* NaCl)NaCl)-value p(* -value(* <(*p (*p 0.05,-value p(*-value (* p<-value -valuep0.05, <-value-value ** 0.05,

Fermentation 2019, 5, 6 4 of 9 Fermentation 2019, 5, x 4 of 9

2.4.2.4. ImpactImpact of of pH pH on onβ-Carotene β-Carotene Production Production of SM14 of SM14 TheThe pH pH of of the the nutrient-reduced nutrient-reduced media media differed differed depending depending on on whether whether seawater seawater or or freshwater freshwater is is used,used, with with values values of of ~5.2, ~5.2, 6.5, 6.5, and and 7.0 7.0 for for 0.1 0.1×× YNBYNB mediamedia inin freshwater,freshwater, 1/3 1/3×× seawater, and seawater, respectively.respectively. PriorPrior workwork hashas shownshown thatthat differencesdifferences inin pHpH havehave potentialpotential impactsimpacts onon carotenoidcarotenoid productionproduction on on microbial microbial producers producers [ 14[14,22].,22]. Thus, Thus,β β-carotene-carotene production production in in freshwater freshwater with with varying varying pHspHs (~5.2, (~5.2, 6.5,6.5, andand 7.0)7.0) waswas quantified.quantified. TheThe resultsresults showedshowed thatthat thethe initialinitial pHpH ofof thethemedia mediadid did not not appearappear to to have have any any significant significant impact impact ononβ β-carotene-carotene productionproduction (Figure(Figure S2a)S2a) oror growthgrowth (Figure(Figure S2b),S2b), andand the the pH pH decreased decreased to to ~4.0 ~4.0 inin allall conditionsconditions beforebefore reaching reaching a a stationary stationary phase phase (Figure (Figure S2b) S2b) when when thethe carotenoids carotenoids started started to to accumulate accumulate in in cells. cells.

2.5.2.5. ImpactImpact of of Carbon-to-Nitrogen Carbon-to-Nitrogen Ratio Ratio (C:N) (C:N) on β-Carotene on β-Carotene Production Production of SM14 of SM14 WeWe previouslypreviously demonstrateddemonstrated thatthat increasingincreasing carbon-to-nitrogencarbon-to-nitrogen (C:N)(C:N) ratioratio fromfrom 8.88.8 toto 5050 improvedimproved carotenoid carotenoid production production of of engineered engineered yeast yeast (including(including SM14)SM14) inin benchtopbenchtop bioreactorsbioreactors [[14].14]. ToTo determine determine if if increasing increasing the the C:N C:N ratio ratio also also increases increases carotenoid carotenoid production production in in nutrient-reduced nutrient-reduced conditions,conditions, we we compared compared the theβ β-carotene-carotene production production of of SM14 SM14 in in 0.1 0.1×× YNB YNBmedia mediawith with the the normal normal C:N C:N −1 ratioratio (8.8) (8.8) (use (use 5 5 g g ammonium ammonium sulfate sulfate L L−1 inin thethe mediamedia asas describeddescribed inin Materials Materials and and Methods) Methods) and and −1 highhigh C:N C:N ratio ratio (50) (50) (use (use 0.88 0.88 g ammoniumg ammonium sulfate sulfate L L−in1 in the the media media while while keeping keeping the the concentration concentration of otherof other constituents constituents the samethe same as described as described in Materials in Mate andrials Methods)and Methods) in freshwater, in freshwater, 1/3× seawater,1/3× seawater, and seawaterand seawater (Figure (Figure3). The results3). The showed results thatshow C:Ned =that 50 significantlyC:N = 50 significantly increased β -caroteneincreased production β-carotene −1 −1 ofproduction SM14 comparing of SM14 with comparing C:N = 8.8,with from C:N 6.51 = 8.8,± 0.37from mg 6.51 g ± 0.37DCW mg to g 9.83−1 DCW± 1.34 to 9.83 mg g± 1.34DCW mg ing−1 −1 −1 freshwater,DCW in freshwater, from 10.70 from± 0.41 10.70 mg ± g 0.41DCW mg g to−1 DCW 11.84 ±to0.36 11.84 mg ± 0.36 g DCWmg g−1 in DCW 1/3× inseawater, 1/3× seawater, and from and −1 −1 8.67from± 8.670.62 ± mg 0.62 g mgDCW g−1 DCW to 10.44 to 10.44± 0.35 ± 0.35 mg mg g g−DCW1 DCW in in seawater seawater after after 72 72 h, h, which which indicates indicates that that increasingincreasing the the C:N C:N ratio ratio can can further further improve improve carotenoid carotenoid production production of of SM14 SM14 in in seawater. seawater.

FigureFigure 3. 3.Effect Effect of of carbon-to-nitrogen carbon-to-nitrogen ratioratio (C:N)(C:N) ononβ β-carotene-carotene productionproduction ofof SM14SM14 afterafter 7272 h. h. FW:FW: freshfresh water; water; SW: SW: seawater.. seawater.. Error Error bars bars are are standard standard deviations. deviations. *: *: Statistically Statistically significantly significantly different different betweenbetween condition condition A A and and B B of of the the same same water water sources sources (* (*p -valuep-value < < 0.05). 0.05).

2.6.2.6. LipidLipid Production Production and and Composition Composition BasedBased on transcriptome transcriptome results results comparing comparing the theglobal global gene gene expression expression of carotenoid of carotenoid hyper- hyper-producersproducers to the toparental the parental strains strains from fromour previo our previousus work, work, we found we found that genes that genes related related with withlipid lipidbiosynthesis biosynthesis (e.g., (e.g., CYB5CYB5, ERG27, ERG27, INO1, INO1) were) were differently differently expressed expressed in the in thehyper-producers hyper-producers [12]; [12 the]; thedirection direction of ofgene gene expression expression changes changes pointed pointed to toth thee possibility possibility that that the the hyper-producers hyper-producers may may have have a ahigher higher lipid lipid content. content. Thus, Thus, in in our our follow-up work, we appliedapplied priorprior knowledgeknowledge fromfrom oleaginousoleaginous yeastsyeasts that that showed showed increased increased lipid lipid content content when when cells cells are are grown grown in nitrogen-limitedin nitrogen-limited conditions conditions [31 ][31] to ourto our strains, strains, and and found found an an increased increased carotenoid carotenoid production production when when the the C:N C:N ratio ratio was was increased increased from from 8.88.8 toto 5050 [[14].14]. Using Raman Raman spectroscopy, spectroscopy, we we conf confirmedirmed that that the the improved improved carotenoid carotenoid production production by byincreasing increasing the the C:N C:N ratio ratio to 50 to was 50 wascorrelated correlated with withincreased increased total lipid total [14]. lipid In [14 this]. work, In this lipid work, analysis lipid was carried out with SM14 cultured in different conditions to identify potential impacts of seawater and varying C:N ratio on lipid content and composition of the cell. Samples in freshwater media were

Fermentation 2019, 5, 6 5 of 9

FermentationFermentationFermentationFermentationanalysisFermentation 2019 2019 2019 ,2019 was5, ,5 2019x,, x 5, ,5 carriedx,, x5 , x out with SM14 cultured in different conditions to identify potential5 impacts 5of of5 59 of 9of 59 of9 of 9 seawater and varying C:N ratio on lipid content and composition of the cell. Samples in freshwater collectedcollectedcollected from from from cultures cultures cultures after after after 72 72 h h72 and and h andsamples samples samples in in seawater seawaterin seawater media media media were were were coll coll ectedcollectedected after after after 120 120 h120 h when when h when β β-- β- collectedmediacollected from were from cultures collected cultures after from after 72 cultures h 72 and h and aftersamples samples 72 h in and seawater in samples seawater media in media seawater were were coll media collectedected were after collectedafter 120 120h when afterh when β 120- β h- carotenecarotenecarotene production production production reached reached reached the the highest thehighest highest level level level (based (based (based on on Figure onFigure Figure 1). 1). As1).As show Asshow show in in Figure Figurein Figure 4a, 4a, 4c, 4a,4c, and 4c,and and carotenewhencarotene productionβ-carotene production productionreached reached the reached thehighest highest thelevel highestlevel (based (based level on Figureon (based Figure 1). on As Figure1). Asshow 1show). in As Figurein show Figure in4a, Figure 4a,4c, 4c,and4 a,cand FigureFigureFigure S3a, S3a, S3a,supplementing supplementing supplementing freshwater freshwater freshwater with with with 0.05 0.05 0.05M M NaCl NaClM NaCl or or increasing increasingor increasing the the C:N theC:N C:Nratio ratio ratio from from from 8.8 8.8 to 8.8to 50 50 to in in50 in FigureandFigure S3a, Figure S3a, supplementing S3a,supplementing supplementing freshwater freshwater freshwater with with 0.05 with0.05 M NaCl M 0.05 NaCl Mor NaClincreasing or increasing or increasing the theC:N C:N theratio ratio C:N from ratiofrom 8.8 fromto8.8 50 to 8.8in 50 toin freshwaterfreshwaterfreshwater significantly significantly significantly increased increased increased li lipidpid li concentration,pid concentration, concentration, yield yield yield or or ratio ratioor ratio of of unsaturated unsaturated of unsaturated to to saturated saturated to saturated fatty fatty fatty freshwater50freshwater in freshwater significantly significantly significantly increased increased increased lipid li pidconcentration, lipidconcentration, concentration, yield yield or ratio yieldor ratio of or unsaturated ratioof unsaturated of unsaturated to saturated to saturated to saturated fatty fatty acidsacidsacidsacids comparedcompared compared compared withwith with with C:NC:N C:N C:N == 8.8= 8.8 = 8.8 in8.8in in freshwater.freshwater.in freshwater. freshwater. ThisThis This This resultresult result result suggestssuggests suggests suggests thatthat that that thethe the thehigherhigher higher higher carotenoidcarotenoid carotenoid carotenoid fattyacids acidscompared compared with with C:N C:N= 8.8 = 8.8in infreshwater. freshwater. This This result result suggests suggests that that the the higher carotenoidcarotenoid productionproductionproductionproduction inin in thesethesein these these conditionsconditions conditions conditions maymay may may bebe be relatedberelated related related withwith with with thethe the thehigherhigher higher higher productionproduction production production ofof of totaltotalof total total lipidslipids lipids oror or thetheor the the productionproduction inin thesethese conditionsconditions maymay bebe relatedrelated withwith thethe higherhigher productionproduction ofof totaltotal lipidslipids oror thethe increasedincreasedincreasedincreased unsaturation unsaturation unsaturation unsaturation of of fattyof fatty of fatty fatty acids acids acids acids observed observed observed observed with with with with th the the additionth additione eaddition addition of of NaClof NaCl of NaCl NaCl or or inor inorcreasing increasing increasingcreasing the the the C:N theC:N C:N C:Nratio. ratio. ratio. ratio. increasedincreased unsaturationunsaturation of of fatty fatty acids acids observed observed with with the th additione addition of of NaCl NaCl or or increasing increasing the the C:N C:N ratio. ratio. In InIn In contrast,Incontrast, contrast, contrast, increasingincreasing increasing increasing thethe the theC:NC:N C:N C:N ratioratio ratio ratio inin in seawaterseawaterin seawater seawater diddid did did notnot not not impaimpa impa impactct lipidctlipidct lipid lipid productionproduction production production oror or fattyorfatty fatty fatty acidacid acid acid contrast,In contrast, increasing increasing the C:Nthe ratioC:N inratio seawater in seawater did not did impact not lipidimpa productionct lipid production or fatty acid or saturationfatty acid saturationsaturationsaturationsaturation (Figures (Figures (Figures (Figures 4b,d, 4b,d, 4b,d, 4b,d, and and and andS3b). S3b). S3b). S3b). Corresponding Corresponding Corresponding Corresponding with with with with β β-carotene -caroteneβ β-carotene-carotene production production production production (Figure (Figure (Figure (Figure 3), 3), 3),the 3),the the total thetotal total total (Figuresaturation4b,d, (Figures and Figure 4b,d, S3b). and CorrespondingS3b). Corresponding with βwith-carotene β-carotene production production (Figure (Figure3), the 3), total the lipid total lipidlipidlipidlipid yieldyield yield yield andand and and concentrationconcentration concentration concentration areare are alsoarealso also also higherhigher higher higher inin in 1/3× 1/3×in 1/3× 1/3× seawaterseawater seawater seawater thanthan than than inin in seawater,seawater,in seawater, seawater, whichwhich which which providesprovides provides provides yieldlipid andyield concentration and concentration are also are higher also inhigher 1/3× inseawater 1/3× seawater than in than seawater, in seawater, which provideswhich provides further furtherfurtherfurtherfurther support support support support that that that thatβ β-carotene-carotene β β-carotene-carotene production production production production is is related isrelated is related related with with with with lipid lipid lipid lipid content. content. content. content. It It has Ithas It has beenhas been been been reported reported reported reported that that that thatsalt salt salt salt supportfurther support that β-carotene that β-carotene production production is related is related with lipid with content. lipid content. It has been It has reported been reported that salt that stress salt stressstressstressstress affects affects affects affects fatty fatty fatty fatty acid acid acid acid composition composition composition composition in in yeast,in yeast, in yeast, yeast, and and and andan an anincrease increasean increase increase in in totalin total in total total sterol sterol sterol and and and andsqualene squalene squalene biosynthesis biosynthesis biosynthesis biosynthesis affectsstress affects fatty acid fatty composition acid composition in yeast, in yeast, and an and increase an increase in total in steroltotal sterol and squalene and squalene biosynthesis biosynthesis was waswaswas wasobserved observed observed observed in in S.in S.in cerevisiae S. cerevisiaeS. cerevisiae cerevisiae under under under under osmotic osmotic osmotic osmotic stress stress stress stress [32]. [32]. [32]. [32]. Squalene Squalene Squalene Squalene is is produced is producedis produced produced via via via the viathe the mevalonate themevalonate mevalonate mevalonate observedwas observed in S. cerevisiaein S. cerevisiaeunder osmoticunder osmotic stress [stress32]. Squalene [32]. Squalene is produced is produced via the via mevalonate the mevalonate (MVA) (MVA)(MVA)(MVA)(MVA) pathway pathway pathway pathway in in yeast,in yeast,in yeast, yeast, and and and andthe the the precursor, theprecursor, precursor, precursor, farnes farnes farnes farnesylyl diphosphate yldiphosphateyl diphosphate diphosphate (FPP) (FPP) (FPP) (FPP) is is also is alsois also alsothe the the precursor theprecursor precursor precursor for for for for pathway(MVA) pathway in yeast, in and yeast, the precursor,and the precursor, farnesyl diphosphate farnesyl diphosphate (FPP) is also (FPP) the precursoris also the for precursor carotenoid for carotenoidcarotenoidcarotenoidcarotenoid biosynthesis biosynthesis biosynthesis biosynthesis [10]. [10]. [10]. [10]. Therefore, Therefore, Therefore, Therefore, an an anupregulation upregulationan upregulation upregulation in in thein the in the MVA theMVA MVA MVA pathway pathway pathway pathway in in responsein response in response response to to osmoticto osmotic to osmotic osmotic biosynthesiscarotenoid biosynthesis [10]. Therefore, [10]. anTherefore, upregulation an upregulation in the MVA in pathway the MVA in pathway response in to response osmotic stressto osmotic may stressstressstressstress maymay may may contributecontribute contribute contribute toto to thetheto the theincreasedincreased increased increased productionproduction production production ofof of carotenoids carotenoidsof carotenoids carotenoids inin in seawater seawaterin seawater seawater andand and and inin in freshwater freshwaterin freshwater freshwater contributestress may to contribute the increased to the production increased of production carotenoids of in carotenoids seawater and in inseawater freshwater and supplemented in freshwater supplementedsupplementedsupplementedsupplemented with with with with additional additional additional additional NaCl. NaCl. NaCl. NaCl. withsupplemented additional with NaCl. additional NaCl.

FigureFigureFigureFigure FigureFigure4. 4. Lipid 4.Lipid 4. Lipid 4.Lipid4. content. LipidLipidcontent. content. content. content.content. ( a(a) )(Total aTotal()a ()Totala( aTotal) )lipid Total lipidTotal lipid lipid concentration lipid concentrationlipid concentration concentration concentration concentration ( ( ( )( ( )and and( ) ) )and andyieldand yield ) andyield yieldyield ( (yield ( () ( )of of ) )( SM14 of )ofSM14 of SM14 )SM14 ofSM14 in SM14in infreshwater, infreshwater, in freshwater, freshwater, infreshwater, freshwater, ( b(b ()b )( )b( totalb) )( b) totaltotaltotal totallipid lipidlipidtotal lipid lipid concentration concentration concentrationlipid concentration concentration concentration ( ( ( ( )) ( )and andand )( )and andyield yield )yield andyield yield ( ( (yield () )( of )of of( ) SM14 SM14 )ofSM14 of )SM14 ofSM14 in in SM14in seawater, seawater, inseawater, in seawater, inseawater, seawater, ( c( )c()c the )(the cthe()c ratio) the( ratio thecratio) ratiothe ofratio of unsaturatedofratio unsaturated ofunsaturated of unsaturated ofunsaturated unsaturated fatty fatty fatty fatty acids fatty fatty to acidsacidsacidsacids to saturatedtoacids saturated tosaturated to saturated tosaturated saturated fatty fatty fatty fatty acids fatty acids acids fatty acids inacids in freshwater,inacids freshwater, infreshwater, in freshwater, infreshwater, freshwater, (d () d( thed) )(the d(thed) ratio ) the(ratio dtheratio) ratiothe of ratio of unsaturatedof ratio unsaturated ofunsaturated of unsaturated ofunsaturated unsaturated fatty fatty fatty fatty acids fatty acids acids fatty acids toacids to saturatedtoacids saturated tosaturated to saturated tosaturated saturated fatty fatty fatty fatty acids fatty fatty in acidsacidsacidsacids in seawater.inacids seawater. inseawater. in seawater. inseawater. seawater. FW: FW: FW: freshFW: FW:fresh fresh FW: fresh water;fresh water; water;fresh water; water; FW FWwater; FW +FW NaCl:+FW + NaCl: FWNaCl:+ +NaCl: NaCl: fresh+ fr NaCl:fresh eshfr waterfresh water esh waterfr eshwater water with with waterwith with 0.05 with 0.05 0.05with M0.05 0.05M NaCl;M 0.05 NaCl;M NaCl;M NaCl; MNaCl; SW: SW: NaCl;SW: seawater.SW: SW:seawater. seawater. SW: seawater. seawater. seawater. Error Error Error Error barsError Error are barsbarsbars barsare arestandardbars arestandard arestandard standardare standard standard deviations. deviations. deviations. deviations. deviations. deviations. *: Statistically*: *: Statistically Statistically*: *: Statistically Statistically*: Statistically significantly signif signif signif significantly icantlysignificantly differenticantly icantlydifferent different different different from different from from C:N from from C:N = C:Nfrom 8.8 C:N C:N= in= 8.8 C:N 8.8= freshwater = 8.8in in8.8 = freshwater in8.8freshwater in freshwater infreshwater (freshwatera,c) or(a,c) (a,c) seawater (a,c) (a,c) (a,c) oror seawater orseawateror seawater( orseawaterb, dseawater) (*(b,d) (b,d)p -value(b,d) (b,d) (* (* (b,d)p (*p-value -value(*

Fermentation 2019, 5, 6 6 of 9 investigated. The carotenoid production of YLH2 and some evolved hyper-producers (SM11, SM12, SM13, SM22, SM24) were quantified in nutrient-reduced condition in either freshwater or 1/3× seawater. As show in Figure S4, consistent with the observation with SM14, the use of 1/3× seawater improved β-carotene production of all tested evolved hyper-producers as well as the ancestor strain YLH2 compared with that in freshwater.

3. Conclusions In conclusion, this work demonstrated that using seawater improved carotenoid production in engineered carotenoids producing S. cerevisiae under nutrient-reduced conditions. This improvement is partially due to the slight osmotic stress imposed by the NaCl content in seawater. Based on our prior transcriptomic results, carotenoid hyper-producers exhibited differential expression in lipid biosynthesis related genes [12]; suggesting a positive correlation between lipid content and β-carotene production. This phenomenon was also observed in this work, as a positive correlation between increased β-carotene production while an increase in total lipid content and unsaturation were observed. Increasing the C:N ratio further improved carotenoid production in both freshwater and seawater, and also was shown to be correlated with lipid content and composition. In comparing the highest carotene production found in this work with previously reported data (see Table S3), the carotene yield we obtained in this work was higher than that reported in the native producer Rhodotorula glutinis [18]. Though the highest carotene production in this work is still lower than our previous work using rich media (YPD) [12] or normal YNB media [14], we showed that using seawater is another potential way to improve carotenoid production, especially under nutrient limited conditions. These findings have the potential to expand the utilization of seawater in microbial fermentation and improve the production of carotenoids.

4. Materials and Methods

4.1. Strains and Growth Conditions Saccharomyces cerevisiae strains SM14, SM11, SM12, SM13, SM22, SM24, and YLH2 [12] were used in this study (Table S2). YLH2 is a S288c strain engineered to produce β-carotene and has the catalase gene ctt1 deleted; it is the parental strain for SM11, SM12, SM13, SM14, SM22, and SM24, which were evolved under oxidative stress to improve β-carotene production [12]. Unless otherwise noted, stock cultures were kept at −80 ◦C, strains were streaked out on YNB plates from frozen stocks and single colonies were picked and cultured aerobically in test tubes containing 3 mL yeast nitrogen base (YNB, 1.7 g L−1, AMRESCO) or 0.1× yeast nitrogen base (0.1× YNB, 0.17 g L−1) media supplemented with 5 g ammonium sulfate L−1 and 20 g glucose L−1 made with either freshwater or synthetic seawater (RICCA) at 30 ◦C. Inoculum of all experiments was collected from overnight cultures in YNB and washed twice with sterile water before inoculation to avoid the impacts of remaining nutrients in the inoculum. The initial OD600 in all experiments was normalized to about 0.1. All experiments were performed in triplicates and statistical tests for significance were determined via 2-tailed Student’s t-test.

4.2. Carotenoid Quantification β-Carotene was quantified by measuring absorbance at 454 nm with a microplate reader (TECAN Infinites M200) [12,14] or by using high-performance liquid chromatography (HPLC, Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) and Agilent ZOBAX Eclipse Plus C18 column (4.6 × 100 mm, 3.5-Micron) as previously described [33]. Briefly, acetonitrile–methanol–isopropanol (50:30:20 v/v) was used as mobile phase at a flow rate of 1 mL min−1, the column temperature was set at 40 ◦C, and β-carotene was detected by UV detector (VWD signal) with wavelength of 450 nm. The quantification by HPLC and absorbance were compared and were found to correlate well (Table S4). The simpler spectrophotometric assay was used for carotenoid quantification in this work. Fermentation 2019, 5, x 3 of 9

Fermentation 2019, 5, x Fermentation 2019, 5, x 5 of 9 5 of 9 collected from cultures after 72 h and samples in seawater media were collcollectedected after from 120 cultures h when after β- 72 h and samples in seawater media were collected after 120 h when β- carotene production reached the highest level (based on Figure 1). As caroteneshow in Figureproduction 4a, 4c, reached and the highest level (based on Figure 1). As show in Figure 4a, 4c, and Figure S3a, supplementing freshwater with 0.05 M NaCl or increasing theFigure C:N ratio S3a,Fermentation fromsupplementing 8.8 2019 to ,50 5, xin freshwater with 0.05 M NaCl or increasing the C:N ratio from 8.8 to 50 in 5 of 9 freshwater significantlyFermentation increased 2019, 5, x li pid concentration, yield or ratio of unsaturatedfreshwater to significantly saturated fatty increased lipid 5concentration, of 9 yield or ratio of unsaturated to saturated fatty acids compared with C:N = 8.8 in freshwater. ThisFigure result 1. suggests Growth kineticsthatacids the comparedof collectedhigherSM14 in carotenoid with0.1×from yeast C:Ncultures nitr= 8.8ogen after in base 72freshwater. h (YNB) and samples media This (20 inresult mL)seawater madesuggests mediawith that were the coll higherected aftercarotenoid 120 h when β- freshwater ( ), 1/3× seawater ( ), and seawater ( ) in 125 mL flasks. (a) growth kinetics production in thesecollected conditions from culturesmay be afterrelated 72 hwith and samplesthe higher in seawaterproductionproduction media of carotenetotal were in lipids thesecoll productionected orconditions the after reached120 may h when bethe related βhighest- with level the (based higher on productionFigure 1). Asof showtotal lipidsin Figure or the4a, 4c, and (DCW: dry cell weight). (b) β-carotene production. increased unsaturationcarotene of fattyproduction acids observed reached withthe highest the addition level of(based NaCl on increasedor FigureincreasingFigure unsaturation1). Asthe S3a, show C:N supplementing ratio. inof Figurefatty acids 4a, freshwater observed4c, and with with th 0.05e addition M NaCl of or NaCl increasing or increasing the C:N the ratio C:N from ratio. 8.8 to 50 in In contrast, increasingFigure S3a,the C:Nsupplementing ratio in seawater freshwater did with not 0.05impa Mct NaCllipidIn or productioncontrast, increasingfreshwater increasing orthe fatty C:N significantly ratio acidthe fromC:N increased ratio8.8 to in50 seawater inlipid concentration, did not impa yieldct orlipid ratio production of unsaturated or fatty to saturated acid fatty 2.3. Impact of NaCl on β-Carotene Production of SM14 saturation (Figuresfreshwater 4b,d, and significantly S3b). Corresponding increased liwithpid concentration,β-carotene production yieldsaturation or ratio (Figureacids (Figures of unsaturatedcompared 3), the 4b,d, total andwith to saturatedS3b). C:N Corresponding = 8.8fatty in freshwater. with β -caroteneThis result production suggests (Figurethat the 3), higher the total carotenoid lipid yield and concentrationacids compared are withalso higherC:N = in8.8 1/3× in It freshwater.seawater has been than reportedThis in lipidresult seawater, that yield suggeststheproduction additionwhichand concentrationthat provides of inthe NaClthese higher increasedconditions are carotenoid also carotenoidhigher may bein related1/3×production seawater with in the somethan higher innative seawater, production which of total provides lipids or the further support thatproduction β-carotene in productionthese conditions is related mayproducers with be lipidrelated [19 content.–23]. with Sodium Itthefurther has higher beenchloride supportincreased reportedproduction is that the unsaturation that β -carotenemain of salt total component lipids productionof fatty or acids inthe isseawater relatedobserved with(the with lipidsynthetic the content. addition seawater It of has NaCl been or reported increasing that the salt C:N ratio. stress affects fattyincreased acid composition unsaturation in yeast, of fatty and acids ancontains increase observed ~0.41 in total with M NaCl),sterol the additionstress and thus squaleneaffects the Inof impactNaClcontrast, fatty biosynthesis or acidof in increasingexcesscreasing composition NaCl the the C:Non in C:N cellyeast, ratio. growthratio and in an andseawater increase β-carotene indid total notproduction sterol impa andct lipid squalene production biosynthesis or fatty acid was observed in InS. contrast,cerevisiae increasing under osmotic the C:N stress wasratio [32]. investigated in Squalene seawater in is didfreshwater produced wasnot observedimpa supplementedviasaturationct the lipid in mevalonate S. production (Figurescerevisiae with va 4b,d,rying underor andfatty concentrations osmotic S3b). acid Corresponding stress of [32]. additional Squalene with NaCl β -caroteneis (Figureproduced production via the mevalonate(Figure 3), the total (MVA) pathway saturationin yeast, and (Figures the precursor, 4b,d, and farnesS3b).2). CorrespondingylAll diphosphate concentrations with(FPP) of(MVA) β NaCl-caroteneis also pathwaytestedlipid the production precursoryield significantly in andyeast, for(Figureconcentration and improved the 3), precursor,the β aretotal-carotene also farnes higher productionyl diphosphatein 1/3× of seawater SM14 (FPP) in thanthe is also in seawater,the precursor which for provides carotenoid biosynthesislipid yield [10]. andTherefore, concentration an upregulation arenutrient-reduced also inhigher the MVA in media1/3× pathway seawatercarotenoid after in 72 responsefurther hthan (Figurebiosynthesis in support toseawater, 2b). osmotic Howe [10].that which Therefore,ver,β-carotene the provides growth anproduction upregulation rate and is finalrelated in biomassthe with MVA lipid decreased pathway content. in Itresponse has been to reported osmotic that salt stress may contributefurther to support the increased that β-carotene production productionas theof carotenoidsconcentration is related in with ofseawaterstress NaCl lipid may increasedcontent. stressand contribute in affects Itfreshwater (Figurehas fattybeen to 2athe acidreported ), suggestingincreased composition that saltproductionthat in whileyeast, NaClofand carotenoids an had increase a detrimental inin totalseawater sterol and and insqualene freshwater biosynthesis supplemented withstress additional affects fatty NaCl. acid compositionimpact in yeast, on cell and growth, an increase supplementedit had in a total positivewas sterol observed with impact and additional squalenein on S. β -carotenecerevisiae NaCl. biosynthesis production. under osmotic stress [32]. Squalene is produced via the mevalonate was observed in S. cerevisiae under Asosmotic the carotenoid stress [32]. hyperproducers Squalene(MVA) is produced pathway were selected via in theyeast, in mevalonate the and presence the precursor, of hydrogen farnes peroxide,yl diphosphate it was (FPP) is also the precursor for (MVA) pathway in yeast, and thenot precursor, surprising farnes that genesyl diphosphate regulatedcarotenoid by(FPP) the isbiosynthesismajor also rethegulators precursor [10]. involvedTherefore, for in an oxidative upregulation stress in response the MVA in pathway in response to osmotic carotenoid biosynthesis [10]. Therefore,yeast (i.e., an upregulation Yap1 [24–26], in Skn7 the MVA [26],stress pathwayMga2 may [27], contribute in andresponse Msn2/4 to tothe osmotic [2 increased8]) were differentially production ofexpressed carotenoids [12]. in seawater and in freshwater stress may contribute to the increasedPrior studies production have shown of carotenoids that exposuresupplemented in seawater to osmotic with and st additionalress in freshwaterconfers NaCl. tolerance to oxidative stress in yeast supplemented with additional NaCl.[29]; suggesting that the same pathways involved in osmotic and oxidative stress resistance overlap. Indeed, the regulators Msn2, Msn4, Pdr3, and Hsf1 have been found to be common between oxidative and osmotic stress [30]. However, it is unclear whether increased oxidative stress tolerance enhances Fermentationcarotenoid2019, 5, 6 production. Thus, it is not known at this point whether the commonalities between7 of 9 the osmotic and oxidative stress tolerance regulon are directly connectedFermentation with the 2019carotenoid, 5, x hyper- 3 of 9 Fermentation 2019, 5, x 3 of 9 4.3. Lipidproducing Quantification phenotype in SM14. The total lipids of S. cerevisiae were extracted in the form of fatty acid methyl ester (FAME) using the sulfuric acid–methanol method as previously described [34], details can be found in Supplementary method. The lipid composition was analyzed by GC–MS using a SHIMADZU-QP2010 SE GC-MS (SHIMADZU CORPORATION, Japan) equipped with a ZB-5MSi column (thickness 0.25 µm; length 30 m; diameter 0.25 mm) as previously described [34].

Supplementary Materials: The following are available online at http://www.mdpi.com/2311-5637/5/1/6/s1, Supplementary Method: Lipid extraction; Table S1: Concentration of common components in normal YNB and synthetic seawater; Table S2: List of strains used; Table S3: Highest β-carotene production in different conditions; Table S4: Comparison of β-carotene measurement between spectrophotometric assay and HPLC; Figure S1: Effect Figure 4. Lipid content. (a) Total lipid concentrationof YNB concentration ( ) and onyield biomass ( ) of formation SM14Figure in andfreshwater, 4.β Lipid-carotene content. (b) production (a) Total of lipid SM14 concentration in freshwater ( ( ) ) andand seawateryield ( ) of SM14 in freshwater, (b) total lipid concentration ( ) and yield (( ) ) afterof SM14 72 h in incubation. seawater, (a) (c) Biomass the ratio (DCW: oftotal unsaturated lipid dry cellconcentration weight). fatty (b) (β-carotene ) and yield concentration. ( ) of SM14 (c) inβ-carotene seawater, yield. (c) the ratio of unsaturated fatty acids to saturated fatty acids in freshwater,Error (d) barsthe ratio areFigurestandard of unsaturated 2. Effect deviations; of fatty NaCl acidsFigure supplementationacids toS2 saturated to: βsaturated-carotene fatty on fatty the production growthacids in kinetics afterfreshwater, 120 and h (a),β(d-carotene) cellthe ratio growthFigure production of unsaturated (solid 1. Growth lines)of SM14 fattykinetics acids of to SM14 saturated in 0.1× fatty yeast nitrogen base (YNB) media (20 mL) made with acids in seawater. FW: fresh water; FW +(DCW:Figure NaCl: dryfr1.esh Growth cell waterin weight) 0.1× kinetics with YNB and 0.05 media of pH MSM14 changeNaCl; in freshwater. in SW:acids (dashed0.1× seawater. yeastin (seawater. lines)a) nitrgrowth Error (b)ogen ofFW: kinetics SM14base fresh (YNB) in (DCW: water; freshwater media dry FW cell + with(20 NaCl: weight) mL) different fr madeesh with freshwaterwater initial with 0 M with pH,( ( (0.05 ), 0.05 )M ), M 1/3×NaCl; seawater SW: seawater. ( ), Errorand seawater ( ) in 125 mL flasks. (a) growth kinetics bars are standard deviations. *: Statisticallyinitialfreshwater signif pHicantly 5.2, ( ( different ), ) ),1/3× initial0.137 seawater from pHM ( 6.5,C:N ( ), =0.2 8.8 )),barsM initialinand ( freshwater are seawater pH ),standard Figure0.3 6.9. M Error(a,c) (4. deviations. Lipid bars ), ) in0.41 content. are125 M standard*: mL( Statistically (flasks.a ) andTotal deviations; (0.5a lipid) signif growthM (concentrationicantlyFigure )kinetics (DCW:NaCl. different S3 ( Individualdryb () β cell-carotene from ) and weight). C:Nyield = ((8.8b) βin ) -caroteneof freshwater SM14 inproduction. freshwater, (a,c) (b) or seawater (b,d) (*Figure p-value 4. < Lipid 0.05, **content. p-valuefatty(DCW: (a <) acidTotal0.01). dry yield. lipidcellyield weight). (a)concentration ( () C:N )( band) β =-carotene 8.8 concentration( in ) freshwater,and production.or yield seawater ( ( ( total )) ) after C:N(b,d)of lipidSM14 = 72(* 50 pconcentration h.-value in Error freshwater, < 0.05,bars **( (are( bp))-value ) standard C:Nand =yield < 8.8 0.01). indeviations. ( freshwater ) of SM14 *: withStatistically in seawater, 0.05 (c) the ratio of unsaturated fatty total lipid concentrationM NaCl,( ) and ( )yield C:Nsignificantly =( 50 )in of freshwater SM14 different in seawater, withfrom 0.05 the Mcontrol(c) NaCl; theacids ratio(no (b)to NaCl) (saturatedof )unsaturated C:N (* p = -valuefatty 8.8 in acids fatty< 1/3 0.05,× inseawater, ** freshwater, p-value2.3. ( < Impact )0.01).(d C:N) the = 50ratioof inNaCl 1/3of unsaturated× on β-Carotene fatty acidsProduction to saturated of SM14 fatty acids to saturated2.3. fatty Impactseawater, acids inof (freshwater, NaCl) C:N on = 8.8β (-Carotened in) the seawater, ratio Production of ( unsaturated) C:N = of 50acids fattySM14 in seawater. inacids seawater. to Error saturated FW: bars fresh arefatty standard water; FW deviations. + NaCl: *:fresh Statistically water with 0.05 M NaCl; SW: seawater. Error 2.7. The Impact of Seawater on β-Carotene Production of Other Evolved 2.7.Hyper-Producers The Impact of Seawater on β-Carotene ProductionIt ofhas Other been Evolvedreported Hyper-Producers that the addition of NaCl increased carotenoid production in some native acids in seawater. FW:significantly fresh water; differentFW + NaCl: from fr C:Nesh water = 8.8 inwith freshwater 0.05 Mbars NaCl; (a) are or standardSW: seawater seawater. deviations. (b) (* Errorp-value *: Statistically <0.05, ** p-value significantly <0.01). different MT: from C:N = 8.8 in freshwater (a,c) Due to potential differences in regulationMethylIt has andbeen tetradecanoate; metabolism, reported that 9-HA: the the same 9-Hexadecenoic addition factorsDue of may toNaCl acid; potential not HA:increased have Hexadecanoic differences the carotenoid in acid; regulation production 11-OA: 11-Octadecenoic andproducers in metabolism,some native [19 acid;–23]. the MS: Sodiumsame factors chloride may is not the have main the component in seawater (the synthetic seawater bars are standard deviations. *: Statistically significantly different fromor C:N seawater = 8.8 in (b,d) freshwater (* p-value (a,c) < 0.05, ** p-value < 0.01). producersMethyl [19 stearate;–23]. FigureSodium S4 :chlorideβ-carotenesame is productionthe impact main in ofcomponentdifferent carotenogenic strain in strainsseawaterbackgrounds. in 0.1(the× YNB Insyntheticdeed,contains with some freshwater seawater ~0.41 reports M ( NaCl),showed) thus that theaddition impact of ofmoderate excess NaCl on cell growth and β-carotene production same impact in differentor seawaterstrain backgrounds. (b,d) (* p-value In

Fermentation 2019, 5, 6 8 of 9

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